Fluoxetine inhibits calcium-activated currents of salamander rodphotoreceptor somata and presynaptic terminals via modulation ofintracellular calcium dynamics

摘要

Purpose: In order to isolate voltage-gated calcium currents in rodsretaining intact axons and presynaptic terminals, it is first necessaryto identify specific blockers of the large calcium-dependent chloridecurrent, ICl(Ca), which obscures them. Based upon previous reports ofits efficacy as an inhibitor of a volume regulated chloride channel(VRAC), a calcium-dependent chloride channel, and the cystic fibrosistransmembrane conductance regulator (CFTR), we investigated whether theserotonin reuptake inhibitor, fluoxetine hydrochloride, could act as aspecific blocker for ICl(Ca) in salamander rod photoreceptor terminals,without affecting other aspects of rod physiology.Methods: Intact rod photoreceptors retaining axons and presynapticterminals were enzymatically dissociated from salamander retinae. Underwhole cell voltage clamp, depolarization-induced whole cell currentswere recorded in the presence and absence of fluoxetine (10 and 50 μMpipette concentration) administered via a puffing pipette or in the bath(25 μM). Changes in intracellular free calcium levels were monitoredas changes in fura-2 fluorescence following brief depolarization withhigh K+ (50 and 100 mM) administered via a puffing pipette in thepresence and absence of fluoxetine (4 and 10 μM) in the bath.Results: When puffed onto cells, fluoxetine inhibited ICl(Ca) in adose-dependent fashion (50 μM=96% reduction; 10 μM=14% reduction).In addition to the reduction in amplitude of ICl(Ca), 4 μM fluoxetine(pipette concentration) significantly reduced the duration of ICl(Ca)(48% reduction). Fluoxetine also suppressed the calcium-activatedpotassium current, IK(Ca), to similar extents (50 μM=75% reduction;10 μM=23% reduction) when puffed onto cells. Preincubation of rodswith 25 μM fluoxetine in the bath significantly reduced outwardcurrents at both 0 mV, where ICl(Ca) is negligible because ECl isabout 0 mV and the bulk of the current is carried by IK(Ca), and at +40mV, where the current is a combination of ICl(Ca) and IK(Ca). Parallelcalcium imaging experiments with fura-2 revealed that preincubation ofrods in 10 μM fluoxetine virtually eliminated the normal rise inintracellular free calcium in somatic (99.6% reduction) and terminal(98% reduction) compartments following brief depolarization with highK+ (100 mM pipette concentration). Cells preincubated in 4 μMfluoxetine, a therapeutically relevant concentration, showed smaller butsignificant reductions in Ca2+ elevations in both somatic (66%reduction) and terminal (36% reduction) compartments and even moresignificant reductions in the duration of sustained calcium levels ofthe terminal compartment (50% reduction) following brief depolarizationwith high K+ (50 mM pipette concentration).Conclusions: We conclude that in addition to blocking ICl(Ca),fluoxetine inhibits IK(Ca). We further conclude that the inhibition ofboth of these currents is the consequence of inhibition of the normalsustained elevation in intracellular calcium following depolarizationand initial calcium influx. Combined, the data suggest that fluoxetinemay have multiple sites of action in rod photoreceptors instead ofacting as a specific inhibitor of ICl(Ca).